Cell culture inserts have become a valuable tool in cell biology as they promote growth and differentiation of a variety of cell types. Cell culture inserts can be used to investigate transport, diffusion, uptake, metabolization and secretion of both synthetic and natural compounds. Furthermore, inserts allow the cultivation of complex three-dimensional tissue.
Apart from the scientific value of studying tissue growth and organization in vitro in biological studies, such tissues are of major importance for the development of in vitro assays to assess toxicity and pharmacokinetic parameters of chemicals, cosmetics and pharmaceutical substances.
With this, the number of animal experiments which are performed to assess such parameters can be significantly reduced. Moreover, the use of human cells and tissue cultivated in cell culture inserts yields much more significant results and reduces the rate of “false positives” i.e. the rate of compounds defined as safe or positive but where a correlation of animal and human data is not possible. Often such false positives are identified only late in drug development or even in clinical trials causing high investments which have to be written off.
Inserts typically consist of a hollow body, closed at one end with a membrane, on which the cells are grown. The membranes are either permeable or, if impermeable, contain microperforations to ensure transport of nutrients across the membrane. The insert is positioned into the well of a cell culture plate in a way that the membrane is in contact with the cell culture medium, either submerged or at the media/air interface.
Cells are usually seeded on the side of the membrane which is showing away from the well of the plate. To allow free diffusion of the cell culture medium, such inserts are positioned into the well plate standing on small feet thus creating a space between the lower part of the insert and the bottom of the well plate. Alternatively, a hanging device can be used, having flanges protruding sideways from the upper part of the insert structure.
The possibility to culture cells on both sides of the insert membrane has gained more attention during the recent years. Cell-to-cell communication across the insert membrane, chemotaxis and other cell migration phenomena can be studied. Electrophysiological analyses are possible on either side of the insert membrane. Apical and basolateral regions of polarized cells can be investigated separately. Stem cells can be separated from feeder cells by cultivation on both sides of the membrane. However, the types of inserts specifically developed for such applications are difficult to use and show significant disadvantages in their practical handling in the cell culture laboratory.
One such insert structure is disclosed in U.S. Pat. No. 5,470,743A. This document describes an assembly consisting of an inverted standard insert on which a support device is positioned. Both support device and insert are connected via a gasket which leaves an inner space consisting of the outer surface of the membrane of the inverted insert on which cells can be grown. After successful cultivation, the assembly is taken apart and the insert with the cells cultivated on the outer surface of the membrane can be hanged into the well of a cell culture plate. Subsequently, cells can be cultured on the inner surface of the membrane. The assembly has to be protected against contamination by a sealing on its upper and lower end. Furthermore, this document discloses a gasket comprising a double sided adhesive to keep support device and insert closely connected during the initial cell culture step. This is a major disadvantage for the disassembly of the parts as there is the risk that the membrane and/or the cells will be damaged during this step. Moreover, the assembly can be used only as a stand-alone device, not in connection with cell culture plates impairing an effective handling especially when many of such cultures are performed at the same time.
Another solution for cell cultivation on both sides of the membrane of an insert is provided in U.S. Pat. No. 5,759,851A. This document describes a movable frame which is placed inside a cylindrical body. A precise positioning of said movable frame within the tubular structure is possible e.g. by using a jig having a flange at one end which exceeds the diameter of the tubular body. The movable frame carries a membrane for the cultivation of biological material. Cultivation on both sides of the membrane is possible by inverting the cylindrical body. To avoid leakage of cell culture medium, the movable frame bears a sealing around its outer diameter. This solution has the disadvantage that a separate tool is needed for height adjustment of the movable frame. Another disadvantage is that it is mandatory to have a firm rim structure or border upon which the jig exerts its action and which, at the same time, serves as a carrier for the membrane. If cells are seeded on such a structure, they grow either on the free, non-supported membrane or on the membrane supported by the rim structure or, in the worst case, on the rim structure itself. Another structure which will come in unwanted direct contact with cells and the culture medium is the sealing of the movable frame. It is obvious that the growth conditions on these different surfaces deviate substantially. Both the movable frame and the sealing ring are artificial materials which influence cell growth, cell proliferation and/or cell differentiation. It can also not be guaranteed that the sealing ring between the frame and the inner wall of the cylindrical body is leak-proof especially when it is shifted by the jig. All these factors make a safe and easy handling difficult and obviate many possible applications.
U.S. Pat. No. 5,759,851A further describes a solution to suppress the formation of air bubbles in the standing insert. This is achieved by forming the end of the movable frame which is pointing towards the bottom of the well plate in a shallow angle. The user then has to carefully lower the device at a defined angle into the cell culture medium and watch for air bubble entrapment. This angular form obviously requires a broader frame; the free-standing part of the membrane which can be used for cell culture is significantly reduced. Furthermore, the handling is difficult. Finally, in most cases inserts are formed in a way that their outer walls fit with minimal distance to the wall of the cell culture plate. It is obvious that the use of a frame with shallow angles is only possible in a well plate which has a very large inner diameter compared to the outer diameter of the insert. Only in this case it is possible to tilt the insert in a way that the angle of the frame and the level of the medium are the same and no air is entrapped.